Stabilized pre-fusion RSV F proteins

ABSTRACT

Stable pre-fusion respiratory syncitial virus (RSV) F proteins, immunogenic compositions including the proteins and uses thereof for the prevention and/or treatment of RSV infection are described.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Section 371 of International Application No.PCT/EP2017/062875, filed May 29, 2017, which was published in theEnglish language on Dec. 7, 2017 under International Publication No. WO2017/207480 A1, and claims priority under 35 U.S.C. § 119(b) to EuropeanApplication No. 16172008.1, filed May 30, 2016, the disclosures of whichare incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

This application contains a sequence listing, which is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “Sequence Listing 688097_559US”, creation date of Nov. 29,2018, and having a size of 30.9 KB. The sequence listing submitted viaEFS-Web is part of the specification and is herein incorporated byreference in its entirety.

The present invention relates to the field of medicine. The invention inparticular relates to recombinant pre-fusion RSV F proteins, to nucleicacid molecules encoding the RSV F proteins, and uses thereof, e.g. invaccines.

BACKGROUND OF THE INVENTION

After discovery of the respiratory syncytial virus (RSV) in the 1950s,the virus soon became a recognized pathogen associated with lower andupper respiratory tract infections in humans. Worldwide, it is estimatedthat 64 million RSV infections occur each year resulting in 160,000deaths (WHO Acute Respiratory Infections Update September 2009). Themost severe disease occurs particularly in premature infants, theelderly and immunocompromised individuals. In children younger than 2years, RSV is the most common respiratory tract pathogen, accounting forapproximately 50% of the hospitalizations due to respiratory infections,and the peak of hospitalization occurs at 2-4 months of age. It has beenreported that almost all children have been infected by RSV by the ageof two. Repeated infection during lifetime is attributed to ineffectivenatural immunity. In the elderly, the RSV disease burden is similar tothose caused by non-pandemic influenza A infections.

RSV is a paramyxovirus, belonging to the subfamily of pneumovirinae. Itsgenome encodes for various proteins, including the membrane proteinsknown as RSV Glycoprotein (G) and RSV fusion (F) protein which are themajor antigenic targets for neutralizing antibodies. Antibodies againstthe fusion-mediating part of the F1 protein can prevent virus uptake inthe cell and thus have a neutralizing effect.

RSV F fuses the viral and host-cell membranes by irreversible proteinrefolding from the labile pre-fusion conformation to the stablepost-fusion conformation. Structures of both conformations have beendetermined for RSV F (McLellan J S, et al. Science 342, 592-598 (2013);McLellan J S, et al. Nat Struct Mol Biol 17, 248-250 (2010); McLellan JS, et al. Science 340, 1113-1117 (2013); Swanson K A, et al. Proceedingsof the National Academy of Sciences of the United States of America 108,9619-9624 (2011)), as well as for the fusion proteins from relatedparamyxoviruses, providing insight into the mechanism of this complexfusion machine. Like other type I fusion proteins, the inactiveprecursor, RSV F₀, requires cleavage during intracellular maturation bya furin-like protease. RSV F contains two furin sites, which leads tothree proteins: F2, p27 and F1, with the latter containing a hydrophobicfusion peptide (FP) at its N-terminus. In order to refold from thepre-fusion to the post-fusion conformation, the refolding region 1 (RR1)between residue 137 and 216, that includes the FP and heptad repeat A(HRA) has to transform from an assembly of helices, loops and strands toa long continuous helix. The FP, located at the N-terminal segment ofRR1, is then able to extend away from the viral membrane and insert intothe proximal membrane of the target cell. Next, the refolding region 2(RR2), which forms the C-terminal stem in the pre-fusion F spike andincludes the heptad repeat B (HRB), relocates to the other side of theRSV F head and binds the HRA coiled-coil trimer with the HRB domain toform the six-helix bundle. The formation of the RR1 coiled-coil andrelocation of RR2 to complete the six-helix bundle are the most dramaticstructural changes that occur during the refolding process.

A vaccine against RSV infection is currently not available, but isdesired due to the high disease burden. The RSV fusion glycoprotein (RSVF) is an attractive vaccine antigen it is the principal target ofneutralizing antibodies in human sera. Most neutralizing antibodies inhuman sera are directed against the pre-fusion conformation, but due toits instability the pre-fusion conformation has a propensity toprematurely refold into the post-fusion conformation, both in solutionand on the surface of the virions. As indicated above, crystalstructures have revealed a large conformational change between thepre-fusion and post-fusion states. The magnitude of the rearrangementsuggested that only a portion of antibodies directed to the post-fusionconformation of RSV-F will be able to cross react with the nativeconformation of the pre-fusion spike on the surface of the virus.Accordingly, efforts to produce a vaccine against RSV have focused ondeveloping vaccines that contain pre-fusion forms of RSV F protein (see,e.g., WO20101149745, WO2010/1149743, WO2009/1079796, WO2012/158613).However, these efforts have not yet yielded stable pre-fusion RSV Fproteins that could be used as candidates for testing in humans.

Therefore, a need remains for efficient vaccines against RSV, inparticular vaccines comprising RSV F proteins in the pre-fusionconformation. The present invention aims at providing such stablepre-fusion RSV F proteins for use in vaccinating against RSV in a safeand efficacious manner.

SUMMARY OF THE INVENTION

The present invention provides stable, recombinant, pre-fusionrespiratory syncytial virus (RSV) fusion (F) proteins, i.e. recombinantRSV F proteins that are stabilized in the pre-fusion conformation, andfragments thereof. The RSV F proteins, or fragments thereof, comprise atleast one epitope that is specific to the pre-fusion conformation Fprotein. In certain embodiments, the pre-fusion RSV F proteins aresoluble proteins. In certain embodiments, the RSV F proteins aretrimers. In certain embodiments the RSV F proteins are multimers oftrimeric RSV F proteins. The invention also provides nucleic acidmolecules encoding the pre-fusion RSV F proteins, or fragments thereof,as well as vectors comprising such nucleic acid molecules.

The invention also relates to compositions, preferably immunogeniccompositions, comprising a RSV F protein, a nucleic acid molecule and/ora vector, and to the use thereof in inducing an immune response againstRSV F protein, in particular to the use thereof as a vaccine. Theinvention also relates to methods for inducing an anti-respiratorysyncytial virus (RSV) immune response in a subject, comprisingadministering to the subject an effective amount of a pre-fusion RSV Fprotein, a nucleic acid molecule encoding said RSV F protein, and/or avector comprising said nucleic acid molecule. Preferably, the inducedimmune response is characterized by neutralizing antibodies to RSVand/or protective immunity against RSV. In particular aspects, theinvention relates to a method for inducing anti-respiratory syncytialvirus (RSV) F antibodies in a subject, comprising administering to thesubject an effective amount of an immunogenic composition comprising apre-fusion RSV F protein, a nucleic acid molecule encoding said RSV Fprotein, and/or a vector comprising said nucleic acid molecule.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Purification of protein F with mutations N67I, S215P, T357K,N371Y, and D486N. Superdex200 gel filtration chromatogram of the eluatefrom the ion-exchange column. The arrow indicates the collected peak.

FIG. 2: A) SDS-PAGE analysis of the F N67I, S215P, T357K, N371Y, D486Nprotein sample containing peak from the SEC chromatogram under reducing(R) and non-reducing (NR). The gels are stained with Coomassie BrilliantBlue.

FIG. 3: The protein concentration of purified RSV F protein F N67I,S215P, T357K, N371Y, D486N was measured by Q Octet assay with CR9501 andCR9503 monoclonal antibodies. CR9501 only binds to RSV F in thepre-fusion conformation. CR9503 binds RSV F both in the pre-fusionconformation and the post-fusion conformation. Plotted as Mean±SE.

FIG. 4: Temperature stability of RSV F protein F N67I, S215P, T357K,N371Y, D486N. Melting temperature (Tm ° C.) determined by differentialscanning fluorimetry (DSF) assay with SyproOrange fluorescent dye.Introduction of the T357K and N371Y substitutions increased the Tm ofPRPM by 3.5 degrees to 68.5 degrees.

DETAILED DESCRIPTION OF THE INVENTION

The fusion protein (F) of the respiratory syncytial virus (RSV) isinvolved in fusion of the viral membrane with a host cell membrane,which is required for infection. RSV F mRNA is translated into a 574amino acid precursor protein designated F0, which contains a signalpeptide sequence at the N-terminus (e.g. amino acid residues 1-26 of SEQID NO: 13) which is removed by a signal peptidase in the endoplasmicreticulum. F0 is cleaved at two sites (between amino acid residues109/110 and 136/137) by cellular proteases (in particular furin, orfurin-like)) removing a short glycosylated intervening sequence (alsoreferred to a p27 region, comprising the amino acid residues 110 to 136,and generating two domains or subunits designated F1 and F2. The F1domain (amino acid residues 137-574) contains a hydrophobic fusionpeptide at its N-terminus and the C-terminus contains the transmembrane(TM) (amino acid residues 530-550) and cytoplasmic region (amino acidresidues 551-574). The F2 domain (amino acid residues 27-109) iscovalently linked to F1 by two disulfide bridges. The F1-F2 heterodimersare assembled as homotrimers in the virion.

A vaccine against RSV infection is currently not yet available. Onepotential approach to producing a vaccine is a subunit vaccine based onpurified RSV F protein. However, for this approach it is desirable thatthe purified RSV F protein is in a conformation which resembles theconformation of the pre-fusion state of RSV F protein, which is stableover time and can be produced in sufficient quantities. In addition, fora soluble, subunit-based vaccine, the RSV F protein needs to betruncated by deletion of the transmembrane (TM) and the cytoplasmicregion to create a soluble secreted F protein (sF). Because the TMregion is responsible for membrane anchoring and stability, theanchorless soluble F protein is considerably more labile than thefull-length protein and will readily refold into the post-fusionend-state. In order to obtain soluble F protein in the stable pre-fusionconformation that shows high expression levels and high stability, thepre-fusion conformation thus needs to be stabilized. Because also thefull length (membrane-bound) RSV F protein is metastable, thestabilization of the pre-fusion conformation is also desirable for thefull length RSV F protein, e.g. for any life attenuated or vector basedvaccine approach.

For the stabilization of soluble RSV F, that is cleaved into the F1 andF2 subunit, in the pre-fusion conformation, a fibritin-basedtrimerization domain was fused to the C-terminus of the soluble RSV-FC-terminal end (McLellan et al., Nature Struct. Biol. 17: 2-248-250(2010); McLellan et al., Science 340(6136):1113-7 (2013)). This fibritindomain or ‘Foldon’ is derived from T4 fibritin and was described earlieras an artificial natural trimerization domain (Letarov et al.,Biochemistry Moscow 64: 817-823 (1993); S-Guthe et al., J. Mol. Biol.337: 905-915. (2004)). However, the trimerization domain does not resultin stable pre-fusion RSV-F protein (Krarup et al., Nature Comm. 6:8143,(2015)). Moreover, these efforts have not yet resulted in candidatessuitable for testing in humans.

Recently, we described combinations of several mutations that arecapable of stabilizing the RSV F protein in the pre-fusion conformation(WO2014/174018 and WO2014/202570). Thus, stable pre-fusion RSV Fproteins have been described comprising a mutation of the amino acidresidue on position 67 and/or a mutation of the amino acid residue onposition 215, preferably a mutation of amino acid residue N/T onposition 67 into I and/or a mutation of amino acid residue S on position215 into P. In addition, soluble pre-fusion RSV F proteins have beendescribed comprising a truncated F1 domain, and comprising a mutation ofthe amino acid residue on position 67 and/or a mutation of the aminoacid residue on position 215, preferably a mutation of amino acidresidue N/T on position 67 into I and/or a mutation of amino acidresidue S on position 215 into P, wherein the protein comprises aheterologous trimerization domain linked to said truncated F1 domain.Additional pre-fusion RSV F proteins have been described, wherein theproteins comprise at least one further mutation, such as a mutation ofthe amino acid residue D on position 486 into N.

According to the present invention it has been found that theintroduction of two other mutations, i.e. on positions 357 and 371(numbering according to SEQ ID NO: 13) also stabilizes the protein inthe pre-fusion conformation.

The present invention thus provides recombinant pre-fusion F proteinscomprising a mutation of the amino acid residue on position 215, inparticular a mutation of the amino acid S on position 215 into P(S215P), in combination with a mutation of the amino acids on positions357, in particular a mutation of the amino acid T on position 357 into K(T357K) and a mutation of the amino acid on position 371, in particulara mutation of the amino acid N on position 371 into Y (N371Y).

The present invention thus provides a unique combination of mutations toprovide recombinant stable pre-fusion RSV F proteins, i.e. RSV Fproteins that are stabilized in the pre-fusion conformation, orfragments thereof. The stable pre-fusion RSV F proteins of theinvention, or fragments thereof, are in the pre-fusion conformation,i.e. they comprise (display) at least one epitope that is specific tothe pre-fusion conformation F protein. An epitope that is specific tothe pre-fusion conformation F protein is an epitope that is notpresented in the post-fusion conformation. Without wishing to be boundby any particular theory, it is believed that the pre-fusionconformation of RSV F protein may contain epitopes that are the same asthose on the RSV F protein expressed on natural RSV virions, andtherefore may provide advantages for eliciting protective neutralizingantibodies.

In certain embodiments, the pre-fusion RSV F proteins of the invention,or fragments thereof, comprise at least one epitope that is recognizedby a pre-fusion specific monoclonal antibody, comprising a heavy chainCDR1 region of SEQ ID NO: 1, a heavy chain CDR2 region of SEQ ID NO: 2,a heavy chain CDR3 region of SEQ ID NO: 3 and a light chain CDR1 regionof SEQ ID NO: 4, a light chain CDR2 region of SEQ ID NO: 5, and a lightchain CDR3 region of SEQ ID NO: 6 (hereafter referred to as CR9501)and/or a pre-fusion specific monoclonal antibody, comprising a heavychain CDR1 region of SEQ ID NO: 7, a heavy chain CDR2 region of SEQ IDNO: 8, a heavy chain CDR3 region of SEQ ID NO: 9 and a light chain CDR1region of SEQ ID NO: 10, a light chain CDR2 region of SEQ ID NO: 11, anda light chain CDR3 region of SEQ ID NO: 12 (referred to as CR9502).CR9501 and CR9502 comprise the heavy and light chain variable regions,and thus the binding specificities, of the antibodies 58C5 and 30D8,respectively, which have previously been shown to bind specifically toRSV F protein in its pre-fusion conformation and not to the post-fusionconformation (see WO2012/006596).

In certain embodiments, the recombinant pre-fusion RSV F proteins aretrimeric.

As used throughout the present application nucleotide sequences areprovided from 5′ to 3′ direction, and amino acid sequences fromN-terminus to C-terminus, as custom in the art.

As indicated above, fragments of the pre-fusion RSV F protein are alsoencompassed by the present invention. The fragment may result fromeither or both of amino-terminal (e.g. by cleaving off the signalsequence) and carboxy-terminal deletions (e.g. by deleting thetransmembrane region and/or cytoplasmic tail). The fragment may bechosen to comprise an immunologically active fragment of the F protein,i.e. a part that will give rise to an immune response in a subject. Thiscan be easily determined using in silico, in vitro and/or in vivomethods, all routine to the skilled person.

In certain embodiments, the encoded proteins or fragments thereofaccording to the invention comprise a signal sequence, also referred toas leader sequence or signal peptide, corresponding to amino acids 1-26of SEQ ID NO: 13. Signal sequences typically are short (e.g. 5-30 aminoacids long) amino acid sequences present at the N-terminus of themajority of newly synthesized proteins that are destined towards thesecretory pathway, and are typically cleaved by signal peptidase togenerate a free signal peptide and a mature protein.

In certain embodiments, the proteins or fragments thereof according tothe invention do not comprise a signal sequence.

In certain embodiments, the (fragments of the) pre-fusion RSV F proteinsare soluble. In certain embodiments, the stable pre-fusion RSV Fproteins or fragments thereof according to the invention comprise atruncated F1 domain, and comprise a heterologous trimerization domainlinked to said truncated F1 domain. According to the invention, it wasshown that by linking a heterologous trimerization domain to theC-terminal amino acid residue of a truncated F1 domain, combined withthe stabilizing mutation(s), soluble RSV F proteins are provided thatshow high expression and that bind to pre-fusion-specific antibodies,indicating that the proteins are in the pre-fusion conformation. Inaddition, the RSV F proteins are stabilized in the pre-fusionconformation, i.e. even after processing of the proteins they still bindto the pre-fusion specific antibodies CR9501 and/or CR9502, indicatingthat the pre-fusion specific epitope is retained.

In certain embodiments, the RSV F proteins are multimers of trimeric RSVF proteins. Thus, in some embodiments, the RSV F proteins may comprisean assembly domain for higher order assemblies of trimers.

It is known that RSV exists as a single serotype having two antigenicsubgroups: A and B. The amino acid sequences of the mature processed Fproteins of the two groups are about 93% identical. As used throughoutthe present application, the amino acid positions are given in referenceto the sequence of RSV F protein of subgroup A (SEQ ID NO: 13). As usedin the present invention, the wording “the amino acid at position “x” ofthe RSV F protein thus means the amino acid corresponding to the aminoacid at position “x” in the RSV F protein of SEQ ID NO: 13. Note that,in the numbering system used throughout this application 1 refers to theN-terminal amino acid of an immature F0 protein (SEQ ID NO: 13) Whenanother RSV strain is used, the amino acid positions of the F proteinare to be numbered with reference to the numbering of the F protein ofSEQ ID NO: 13 by aligning the sequences of the other RSV strain with theF protein of SEQ ID NO: 13 with the insertion of gaps as needed.Sequence alignments can be done using methods well known in the art,e.g. by CLUSTALW, Bioedit or CLC Workbench.

In certain embodiments, the RSV strain is the RSV strain of SEQ ID NO:20.

In certain embodiments, the RSV strain is an RSV B strain. In certainembodiments, the RSV strain is the RSV B strain of SEQ ID NO: 15.

An amino acid according to the invention can be any of the twentynaturally occurring (or ‘standard’ amino acids) or variants thereof,such as e.g. D-amino acids (the D-enantiomers of amino acids with achiral center), or any variants that are not naturally found inproteins, such as e.g. norleucine. The standard amino acids can bedivided into several groups based on their properties. Important factorsare charge, hydrophilicity or hydrophobicity, size and functionalgroups. These properties are important for protein structure andprotein-protein interactions. Some amino acids have special propertiessuch as cysteine, that can form covalent disulfide bonds (or disulfidebridges) to other cysteine residues, proline that induces turns of theprotein backbone, and glycine that is more flexible than other aminoacids. Table 1 shows the abbreviations and properties of the standardamino acids.

It will be appreciated by a skilled person that the mutations can bemade to the protein by routine molecular biology procedures. Themutations according to the invention preferably result in increasedexpression levels and/or increased stabilization of the pre-fusion RSV Fproteins as compared RSV F proteins that do not comprise thesemutation(s).

In certain embodiments, the pre-fusion RSV F proteins or fragmentsthereof comprise at least one further mutation selected from the groupconsisting of:

-   -   (a) a mutation of the amino acid residue on position 67; and    -   (b) a mutation of the amino acid residue on position 486.

In certain embodiments, the at least one further mutation is selectedfrom the group consisting of:

-   -   (a) a mutation of the amino acid residue N/T on position 67 into        I; and    -   (b) a mutation of the amino acid residue D on position 486 into        N.

In certain embodiments, the pre-fusion RSV F proteins or fragmentsthereof comprise at least four mutations (as compared to a wild-type RVF protein, e.g. the comprising the amino acid sequence of SEQ ID NO:13). In certain embodiments, the proteins or fragments thereof compriseat least five mutations.

In certain embodiments, the proteins or fragments thereof comprise atleast six mutations.

In certain embodiments, the pre-fusion RSV F polypeptides thus compriseat least one further mutation selected from the group consisting of:

-   -   (a) a mutation of the amino acid residue on position 46;    -   (b) a mutation of the amino acid residue on position 83;    -   (c) a mutation of the amino acid residue on position 92;    -   (d) a mutation of the amino acid residue on position 184;    -   (e) a mutation of the amino acid residue on position 203;    -   (f) a mutation of the amino acid residue on position 207; and    -   (g) a mutation of the amino acid residue on position 487.

In certain embodiments, the at least one further mutation is selectedfrom the group consisting of:

-   -   (a) a mutation of the amino acid residue S on position 46 into        G;    -   (b) a mutation of the amino acid residue L on position 83 into        M:    -   (c) a mutation of the amino acid residue E on position 92 into        D;    -   (d) a mutation of the amino acid residue G on position 184 into        N;    -   (e) a mutation of the amino acid residue L on position 203 into        I;    -   (f) a mutation of the amino acid residue V on position 207 into        I: and    -   (g) a mutation of the amino acid residue E on position 487 into        Q, N or I.

In certain other embodiments, the heterologous trimerization domaincomprises the amino acid sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ IDNO: 14).

As described above, in certain embodiments, the proteins of theinvention or fragments thereof comprise a truncated F1 domain. As usedherein a “truncated” F1 domain refers to a F1 domain that is not a fulllength F1 domain, i.e. wherein either N-terminally or C-terminally oneor more amino acid residues have been deleted. According to theinvention, at least the transmembrane domain and cytoplasmic tail havebeen deleted to permit expression as a soluble ectodomain.

In certain other embodiments, the trimerization domain is linked toamino acid residue 513 of the RSV F1 domain. In certain embodiments, thetrimerization domain thus comprises SEQ ID NO: 14 and is linked to aminoacid residue 513 of the RSV F1 domain.

In certain embodiments, the RSV F protein of the invention comprises theamino acid sequence of SEQ ID NO: 21.

In certain embodiments, the level of expression of the pre-fusion RSV Fproteins of the invention is increased, as compared to a wild-type RSV Fprotein. In certain embodiments the level of expression is increased atleast 5-fold, preferably up to 10-fold. In certain embodiments, thelevel of expression is increased more than 10-fold.

The pre-fusion RSV F proteins according to the invention are stable,i.e. do not readily change into the post-fusion conformation uponprocessing of the proteins, such as e.g. purification, freeze-thawcycles, and/or storage etc.

In certain embodiments, the pre-fusion RSV F proteins according to theinvention have an increased stability upon storage a 4° C. as comparedto a RSV F protein without the mutation(s). In certain embodiments, theproteins are stable upon storage at 4° C. for at least 30 days,preferably at least 60 days, preferably at least 6 months, even morepreferably at least 1 year. With “stable upon storage”, it is meant thatthe proteins still display the at least one epitope specific for the apre-fusion specific antibody (e.g. CR9501) upon storage of the proteinin solution (e.g. culture medium) at 4° C. for at least 30 days. Incertain embodiments, the proteins display the at least one pre-fusionspecific epitope for at least 6 months, preferably for at least 1 yearupon storage of the pre-fusion RSV F proteins at 4° C.

In certain embodiments, the pre-fusion RSV F proteins according to theinvention have an increased stability when subjected to heat, ascompared to RSV F proteins without said mutation(s). In certainembodiments, the pre-fusion REV F proteins are heat stable for at least30 minutes at a temperature of 55° C., preferably at 58° C., morepreferably at 60° C. With “heat stable” it is meant that the proteinsstill display the at least one pe-fusion specific epitope after havingbeen subjected for at least 30 minutes to an increased temperature (i.e.a temperature of 55° C. or above), e.g. as determined using a method asdescribed in the Examples (see FIG. 4).

In certain embodiments, the proteins display the at least one pre-fusionspecific epitope after being subjected to 1 to 6 freeze-thaw cycles inan appropriate formulation buffer.

As used throughout the present application nucleotide sequences areprovided from 5′ to 3′ direction, and amino acid sequences fromN-terminus to C-terminus, as custom in the art.

In certain embodiments, the encoded proteins according to the inventionfurther comprise a leader sequence, also referred to as signal sequenceor signal peptide, corresponding to amino acids 1-26 of SEQ ID NO: 13.This is a short (typically 5-30 amino acids long) peptide present at theN-terminus of the majority of newly synthesized proteins that aredestined towards the secretory pathway. In certain embodiments, theproteins according to the invention do not comprise a leader sequence.

In certain embodiments, the proteins comprise a HIS-Tag. A His-Tag orpolyhistidine-tag is an amino acid motif in proteins that consists of atleast five histidine (H) residues, often at the N- or C-terminus of theprotein, which is generally used for purification purposes.

The present invention further provides nucleic acid molecules encodingthe RSV F proteins according to the invention.

In preferred embodiments, the nucleic acid molecules encoding theproteins according to the invention are codon-optimized for expressionin mammalian cells, preferably human cells. Methods ofcodon-optimization are known and have been described previously (e.g. WO96/09378). A sequence is considered codon-optimized if at least onenon-preferred codon as compared to a wild type sequence is replaced by acodon that is more preferred. Herein, a non-preferred codon is a codonthat is used less frequently in an organism than another codon codingfor the same amino acid, and a codon that is more preferred is a codonthat is used more frequently in an organism than a non-preferred codon.The frequency of codon usage for a specific organism can be found incodon frequency tables, such as in website: www.kazusa.or.jp/codon.Preferably more than one non-preferred codon, preferably most or allnon-preferred codons, are replaced by codons that are more preferred.Preferably the most frequently used codons in an organism are used in acodon-optimized sequence. Replacement by preferred codons generallyleads to higher expression.

It will be understood by a skilled person that numerous differentpolynucleotides and nucleic acid molecules can encode the same proteinas a result of the degeneracy of the genetic code. It is also understoodthat skilled persons may, using routine techniques, make nucleotidesubstitutions that do not affect the protein sequence encoded by thenucleic acid molecules to reflect the codon usage of any particular hostorganism in which the proteins are to be expressed. Therefore, unlessotherwise specified, a “nucleotide sequence encoding an amino acidsequence” includes all nucleotide sequences that are degenerate versionsof each other and that encode the same amino acid sequence. Nucleotidesequences that encode proteins and RNA may or may not include introns.

Nucleic acid sequences can be cloned using routine molecular biologytechniques, or generated de novo by DNA synthesis, which can beperformed using routine procedures by service companies having businessin the field of DNA synthesis and/or molecular cloning (e.g. GeneArt,GenScripts, Invitrogen, Eurofins).

The invention also provides vectors comprising a nucleic acid moleculeas described above. In certain embodiments, a nucleic acid moleculeaccording to the invention thus is part of a vector. Such vectors caneasily be manipulated by methods well known to the person skilled in theart, and can for instance be designed for being capable of replicationin prokaryotic and/or eukaryotic cells. In addition, many vectors can beused for transformation of eukaryotic cells and will integrate in wholeor in part into the genome of such cells, resulting in stable host cellscomprising the desired nucleic acid in their genome. The vector used canbe any vector that is suitable for cloning DNA and that can be used fortranscription of a nucleic acid of interest. Suitable vectors accordingto the invention are e.g. adenovectors, alphavirus, paramyxovirus,vaccinia virus, herpes virus, retroviral vectors etc. The person skilledin the art is capable of choosing suitable expression vectors, andinserting the nucleic acid sequences of the invention in a functionalmanner.

Host cells comprising the nucleic acid molecules encoding the pre-fusionRSV F proteins form also part of the invention. The pre-fusion RSV Fproteins may be produced through recombinant DNA technology involvingexpression of the molecules in host cells, e.g. Chinese hamster ovary(CHO) cells, tumor cell lines, BHK cells, human cell lines such asHEK293 cells, PER.C6 cells, or yeast, fungi, insect cells, and the like,or transgenic animals or plants. In certain embodiments, the cells arefrom a multicellular organism, in certain embodiments they are ofvertebrate or invertebrate origin. In certain embodiments, the cells aremammalian cells. In certain embodiments, the cells are human cells. Ingeneral, the production of a recombinant proteins, such the pre-fusionRSV F proteins of the invention, in a host cell comprises theintroduction of a heterologous nucleic acid molecule encoding theprotein in expressible format into the host cell, culturing the cellsunder conditions conducive to expression of the nucleic acid moleculeand allowing expression of the protein in said cell. The nucleic acidmolecule encoding a protein in expressible format may be in the form ofan expression cassette, and usually requires sequences capable ofbringing about expression of the nucleic acid, such as enhancer(s),promoter, polyadenylation signal, and the like. The person skilled inthe art is aware that various promoters can be used to obtain expressionof a gene in host cells. Promoters can be constitutive or regulated, andcan be obtained from various sources, including viruses, prokaryotic, oreukaryotic sources, or artificially designed.

Cell culture media are available from various vendors, and a suitablemedium can be routinely chosen for a host cell to express the protein ofinterest, here the pre-fusion RSV F proteins. The suitable medium may ormay not contain serum.

A “heterologous nucleic acid molecule” (also referred to herein as‘transgene’) is a nucleic acid molecule that is not naturally present inthe host cell. It is introduced into for instance a vector by standardmolecular biology techniques. A transgene is generally operably linkedto expression control sequences. This can for instance be done byplacing the nucleic acid encoding the transgene(s) under the control ofa promoter. Further regulatory sequences may be added. Many promoterscan be used for expression of a transgene(s), and are known to theskilled person, e.g. these may comprise viral, mammalian, syntheticpromoters, and the like. A non-limiting example of a suitable promoterfor obtaining expression in eukaryotic cells is a CMV-promoter (U.S.Pat. No. 5,385,839), e.g. the CMV immediate early promoter, for instancecomprising nt. −735 to +95 from the CMV immediate early geneenhancer/promoter. A polyadenylation signal, for example the bovinegrowth hormone polyA signal (U.S. Pat. No. 5,122,458), may be presentbehind the transgene(s). Alternatively, several widely used expressionvectors are available in the art and from commercial sources, e.g. thepcDNA and pEF vector series of Invitrogen, pMSCV and pTK-Hyg from BDSciences, pCMV-Script from Stratagene, etc, which can be used torecombinantly express the protein of interest, or to obtain suitablepromoters and/or transcription terminator sequences, polyA sequences,and the like.

The cell culture can be any type of cell culture, including adherentcell culture, e.g. cells attached to the surface of a culture vessel orto microcarriers, as well as suspension culture. Most large-scalesuspension cultures are operated as batch or fed-batch processes becausethey are the most straightforward to operate and scale up. Nowadays,continuous processes based on perfusion principles are becoming morecommon and are also suitable. Suitable culture media are also well knownto the skilled person and can generally be obtained from commercialsources in large quantities, or custom-made according to standardprotocols. Culturing can be done for instance in dishes, roller bottlesor in bioreactors, using batch, fed-batch, continuous systems and thelike. Suitable conditions for culturing cells are known (see e.g. TissueCulture, Academic Press, Kruse and Paterson, editors (1973), and R. I.Freshney, Culture of animal cells: A manual of basic technique, fourthedition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9)).

The invention further provides compositions comprising a pre-fusion RSVF protein and/or a nucleic acid molecule, and/or a vector, as describedabove. The invention thus provides compositions comprising a pre-fusionRSV F protein that displays an epitope that is present in a pre-fusionconformation of the RSV F protein but is absent in the post-fusionconformation. The invention also provides compositions comprising anucleic acid molecule and/or a vector, encoding such pre-fusion RSV Fprotein. The invention further provides immunogenic compositionscomprising a pre-fusion RSV F protein, and/or a nucleic acid molecule,and/or a vector, as described above. The invention also provides the useof a stabilized pre-fusion RSV F protein, a nucleic acid molecule,and/or a vector, according to the invention, for inducing an immuneresponse against RSV F protein in a subject. Further provided aremethods for inducing an immune response against RSV F protein in asubject, comprising administering to the subject a pre-fusion RSV Fprotein, and/or a nucleic acid molecule, and/or a vector, according tothe invention. Also provided are pre-fusion RSV F proteins, nucleic acidmolecules, and/or vectors, according to the invention for use ininducing an immune response against RSV F protein in a subject. Furtherprovided is the use of the pre-fusion RSV F proteins, and/or nucleicacid molecules, and/or vectors according to the invention for themanufacture of a medicament for use in inducing an immune responseagainst RSV F protein in a subject.

The pre-fusion RSV F proteins, nucleic acid molecules, or vectors of theinvention may be used for prevention (prophylaxis) and/or treatment ofRSV infections. In certain embodiments, the prevention and/or treatmentmay be targeted at patient groups that are susceptible RSV infection.Such patient groups include, but are not limited to e.g., the elderly(e.g. ≥50 years old, ≥60 years old, and preferably ≥65 years old), theyoung (e.g. ≤5 years old, ≤1 year old), hospitalized patients andpatients who have been treated with an antiviral compound but have shownan inadequate antiviral response.

The pre-fusion RSV F proteins, nucleic acid molecules and/or vectorsaccording to the invention may be used e.g. in stand-alone treatmentand/or prophylaxis of a disease or condition caused by RSV, or incombination with other prophylactic and/or therapeutic treatments, suchas (existing or future) vaccines, antiviral agents and/or monoclonalantibodies.

The invention further provides methods for preventing and/or treatingRSV infection in a subject utilizing the pre-fusion RSV F proteins,nucleic acid molecules and/or vectors according to the invention. In aspecific embodiment, a method for preventing and/or treating RSVinfection in a subject comprises administering to a subject in needthereof an effective amount of a pre-fusion RSV F protein, nucleic acidmolecule and/or a vector, as described above. A therapeuticallyeffective amount refers to an amount of a protein, nucleic acid moleculeor vector, that is effective for preventing, ameliorating and/ortreating a disease or condition resulting from infection by RSV.Prevention encompasses inhibiting or reducing the spread of RSV orinhibiting or reducing the onset, development or progression of one ormore of the symptoms associated with infection by RSV. Amelioration asused in herein may refer to the reduction of visible or perceptibledisease symptoms, viremia, or any other measurable manifestation ofinfluenza infection.

For administering to subjects, such as humans, the invention may employpharmaceutical compositions comprising a pre-fusion RSV F protein, anucleic acid molecule and/or a vector as described herein, and apharmaceutically acceptable carrier or excipient. In the presentcontext, the term “pharmaceutically acceptable” means that the carrieror excipient, at the dosages and concentrations employed, will not causeany unwanted or harmful effects in the subjects to which they areadministered. Such pharmaceutically acceptable carriers and excipientsare well known in the art (see Remington's Pharmaceutical Sciences, 18thedition, A. R. Gennaro, Ed., Mack Publishing Company [1990];Pharmaceutical Formulation Development of Peptides and Proteins, S.Frokjaer and L. Hovgaard, Eds., Taylor & Francis [2000]; and Handbook ofPharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., PharmaceuticalPress [2000]). The RSV F proteins, or nucleic acid molecules, preferablyare formulated and administered as a sterile solution although it mayalso be possible to utilize lyophilized preparations. Sterile solutionsare prepared by sterile filtration or by other methods known per se inthe art. The solutions are then lyophilized or filled intopharmaceutical dosage containers. The pH of the solution generally is inthe range of pH 3.0 to 9.5, e.g. pH 5.0 to 7.5. The RSV F proteinstypically are in a solution having a suitable pharmaceuticallyacceptable buffer, and the composition may also contain a salt.Optionally stabilizing agent may be present, such as albumin. In certainembodiments, detergent is added. In certain embodiments, the RSV Fproteins may be formulated into an injectable preparation.

In certain embodiments, a composition according to the invention furthercomprises one or more adjuvants. Adjuvants are known in the art tofurther increase the immune response to an applied antigenicdeterminant. The terms “adjuvant” and “immune stimulant” are usedinterchangeably herein, and are defined as one or more substances thatcause stimulation of the immune system. In this context, an adjuvant isused to enhance an immune response to the RSV F proteins of theinvention. Examples of suitable adjuvants include aluminium salts suchas aluminium hydroxide and/or aluminium phosphate; oil-emulsioncompositions (or oil-in-water compositions), including squalene-wateremulsions, such as MF59 (see e.g. WO 90/14837); saponin formulations,such as for example QS21 and Immunostimulating Complexes (ISCOMS) (seee.g. U.S. Pat. No. 5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762,WO 2005/002620); bacterial or microbial derivatives, examples of whichare monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motifcontaining oligonucleotides, ADP-ribosylating bacterial toxins ormutants thereof, such as E. coli heat labile enterotoxin LT, choleratoxin CT, and the like; eukaryotic proteins (e.g. antibodies orfragments thereof (e.g. directed against the antigen itself or CD1a,CD3, CD7, CD80) and ligands to receptors (e.g. CD40L, GMCSF, GCSF, etc),which stimulate immune response upon interaction with recipient cells.In certain embodiments the compositions of the invention comprisealuminium as an adjuvant, e.g. in the form of aluminium hydroxide,aluminium phosphate, aluminium potassium phosphate, or combinationsthereof, in concentrations of 0.05-5 mg, e.g. from 0.075-1.0 mg, ofaluminium content per dose.

The pre-fusion RSV F proteins may also be administered in combinationwith or conjugated to nanoparticles, such as e.g. polymers, liposomes,virosomes, virus-like particles. The pre-fusion F proteins may becombined with, encapsidated in or conjugated to the nanoparticles withor without adjuvant. Encapsulation within liposomes is described, e.g.in U.S. Pat. No. 4,235,877. Conjugation to macromolecules is disclosed,for example in U.S. Pat. Nos. 4,372,945 or 4,474,757. In otherembodiments, the RSV F proteins are assembled in higher order assembliesof multimers.

In other embodiments, the compositions do not comprise adjuvants.

In certain embodiments, the invention provides methods for making avaccine against respiratory syncytial virus (RSV), comprising providinga composition according to the invention and formulating it into apharmaceutically acceptable composition. The term “vaccine” refers to anagent or composition containing an active component effective to inducea certain degree of immunity in a subject against a certain pathogen ordisease, which will result in at least a decrease (up to completeabsence) of the severity, duration or other manifestation of symptomsassociated with infection by the pathogen or the disease. In the presentinvention, the vaccine comprises an effective amount of a pre-fusion RSVF protein and/or a nucleic acid molecule encoding a pre-fusion RSV Fprotein, and/or a vector comprising said nucleic acid molecule, whichresults in an immune response against the F protein of RSV. Thisprovides a method of preventing serious lower respiratory tract diseaseleading to hospitalization and the decrease in frequency ofcomplications such as pneumonia and bronchiolitis due to RSV infectionand replication in a subject. The term “vaccine” according to theinvention implies that it is a pharmaceutical composition, and thustypically includes a pharmaceutically acceptable diluent, carrier orexcipient. It may or may not comprise further active ingredients. Incertain embodiments it may be a combination vaccine that furthercomprises other components that induce an immune response, e.g. againstother proteins of RSV and/or against other infectious agents. Theadministration of further active components may for instance be done byseparate administration or by administering combination products of thevaccines of the invention and the further active components.

Compositions may be administered to a subject, e.g. a human subject. Thetotal dose of the RSV F proteins in a composition for a singleadministration can for instance be about 0.01 μg to about 10 mg, e.g. 1μg-1 mg, e.g. 10 μg-100 μg. Determining the recommended dose will becarried out by experimentation and is routine for those skilled in theart.

Administration of the compositions according to the invention can beperformed using standard routes of administration. Non-limitingembodiments include parenteral administration, such as intradermal,intramuscular, subcutaneous, transcutaneous, or mucosal administration,e.g. intranasal, oral, and the like. In one embodiment a composition isadministered by intramuscular injection. The skilled person knows thevarious possibilities to administer a composition, e.g. a vaccine inorder to induce an immune response to the antigen(s) in the vaccine.

A subject as used herein preferably is a mammal, for instance a rodent,e.g. a mouse, a cotton rat, or a non-human-primate, or a human.Preferably, the subject is a human subject.

The proteins, nucleic acid molecules, vectors, and/or compositions mayalso be administered, either as prime, or as boost, in a homologous orheterologous prime-boost regimen. If a boosting vaccination isperformed, typically, such a boosting vaccination will be administeredto the same subject at a time between one week and one year, preferablybetween two weeks and four months, after administering the compositionto the subject for the first time (which is in such cases referred to as‘priming vaccination’). In certain embodiments, the administrationcomprises a prime and at least one booster administration.

In addition, the proteins of the invention may be used as diagnostictool, for example to test the immune status of an individual byestablishing whether there are antibodies in the serum of suchindividual capable of binding to the protein of the invention. Theinvention thus also relates to an in vitro diagnostic method fordetecting the presence of an RSV infection in a patient said methodcomprising the steps of a) contacting a biological sample obtained fromsaid patient with a protein according to the invention; and b) detectingthe presence of antibody-protein complexes.

Stabilized pre-fusion RSV F proteins obtainable and/or obtained by suchmethod also form part of the invention, as well as uses thereof asdescribed above.

EXAMPLES Example 1 Preparation of Stable Pre Fusion RSV F Polypeptide ofSEQ ID NO: 21

To increase the stability of RSV F in the pre-fusion conformation twoadditional amino acid substitutions were introduced in a pre-fusion RSVF variant that was described previously (WO2014/174018 andWO2014/202570). The constructs were synthesized and codon-optimized atGene Art (Life Technologies, Carlsbad, Calif.). The constructs werecloned into pCDNA2004 or generated by standard methods widely knownwithin the field involving site-directed mutagenesis and PCR andsequenced. The expression platform used was the 293Freestyle cells (LifeTechnologies). The cells were transiently transfected using 293Fectin(Life Technologies) according to the manufacturer's instructions andcultured for 5 days at 37° C. and 10% CO₂. The culture supernatant washarvested and spun for 5 minutes at 300 g to remove cells and cellulardebris. The spun supernatant was subsequently sterile filtered using a0.22 um vacuum filter and stored at 4° C. until use.

Example 2 Purification of Pre-Fusion RSV F Protein

The recombinant polypeptide was purified by a 2-step purificationprotocol applying a cat-ion exchange column for the initial purificationand subsequently a superdex200 column for the polishing step to removeresidual contaminants. For the initial ion-exchange step the culturesupernatant was diluted with 2 volumes of 50 mM NaOAc pH 5.0 and passedover a 5 ml HiTrap Capto S column at 5 ml per minute. Subsequently thecolumn was washed with 10 column volumes (CV) of 20 mM NaOAc, 50 mMNaCl, 0.01% (v/v) tween20, pH 5 and eluted 2 CV of 20 mM NaOAc, 1M NaCl,0.01% (v/v) tween20, pH 5. The eluate was concentrated using a spinconcentrator and the protein was further purified using a superdex200column using 40 mM Tris, 500 mM NaCl, 0.01% (v/v) tween20, pH 7.4 asrunning buffer. In FIG. 1 the chromatogram of the gel filtration columnis shown. The dominant peak contained the pre-fusion RSV F protein. Thefractions containing this peak were again pooled and the proteinconcentration was determined using OD280 and stored a 4° C. until use.In FIG. 2 a non-reduced and reduced SDS-PAGE analysis of the finalprotein preparation is shown and as can be seen the purity was >95%. Theidentity of the band was verified using western blotting and protein Fspecific antibodies (not shown).

Quantitative Octet (BioLayer Interferometry) was used for measuringprotein concentration in the supernatants. CR9501 (an antibodyspecifically recognizing pre-fusion RSV F protein) and CR9503(recognizing post-fusion RSV F protein) were biotinylated by standardprotocols and immobilized on Streptavidin biosensors (ForteBio,Portsmouth, UK). Afterwards, the coated biosensors were blocked in mockcell culture supernatant. A quantitative experiment was performed asfollows: temperature 30 C, shaking speed 1000 rpm, time of the assay 300sec.

The concentration of the protein was calculated using a standard curve.The standard curve was prepared for each coated antibody using thepre-fusion RSV F protein (Krarup et. al., 2015, supra) diluted in mockmedium (FIG. 3). The data analysis was done using the ForteBio DataAnalysis 6.4 software (ForteBio).

Example 3 Temperature Stability of the RSV F Protein

Temperature stability of the purified protein was determined bydifferential scanning fluorometry (DSF). The purified pre-fusion Fprotein was mixed with SYPRO orange fluorescent dye (Life TechnologiesS6650) in a 96-well optical qPCR plate. The optimal dye and proteinconcentration was determined experimentally (data not shown). Proteindilutions were performed in PBS, and a negative control samplecontaining the dye only was used as a reference subtraction. Themeasurement was performed in a qPCR instrument (Applied Biosystems ViiA7) using the following parameters: a temperature ramp from 25-95° C.with a rate of 0.015° C. per second. Data was collected continuously.The melting curves were plotted using GraphPad PRISM software (version5.04). Melting temperatures were calculated at the 50% maximum offluorescence using a non-linear EC50 shift equation. The meltingtemperature of the RSV F protein of SEQ ID NO: 21 was 68.5 degrees (FIG.4). A reference pre-fusion RSV F without substitutions at position 357and 371 had a melting temperature of 65.0 which means the doublemutation increased the melting temperature by 3.5 degrees.

TABLE 1 Standard amino acids, abbreviations and properties Side chainSide chain charge Amino Acid 3-Letter 1-Letter polarity (pH 7.4) alanineAla A non-polar Neutral arginine Arg R polar Positive asparagine Asn Npolar Neutral aspartic acid Asp D polar Negative cysteine Cys Cnon-polar Neutral glutamic acid Glu E polar Negative glutamine Gln Qpolar Neutral glycine Gly G non-polar Neutral histidine His H polarpositive(10%) neutral(90%) isoleucine Ile I non-polar Neutral leucineLeu L non-polar Neutral lysine Lys K polar Positive methionine Met Mnon-polar Neutral phenylalanine Phe F non-polar Neutral proline Pro Pnon-polar Neutral serine Ser S polar Neutral threonine Thr T polarNeutral tryptophan Trp W non-polar Neutral tyrosine Tyr Y polar Neutralvaline Val V non-polar Neutral

TABLE 2  Amino acid sequences of antibodies CR9501 and CR9502 AbVH domain VH CDR1 VH CDR2 VH CDR3 CR9501 Amino acids 1-125 GASINSDNYYWTHISYTGNTYYTPSLKS CGAYVLISNCGWFDS  of SEQ ID NO: 16 (SEQ ID NO: 1)(SEQ ID NO: 2) (SEQ ID NO: 3) CR9502 Amino acids 1-121 GFTFSGHTIAWVSTNNGNTEYAQKIQG EWLVMGGFAFDH  of SEQ ID NO:18 (SEQ ID NO: 7)(SEQ ID NO: 8) (SEQ ID NO: 9) CR9501 Amino acids 1-107 QASQDISTYLNGASNLET QQYQYLPYT of SEQ ID NO: 17 (SEQ ID NO: 4) (SEQ ID NO: 5)(SEQ ID NO: 6) CR9502 Amino acids 1-110 GANNIGSQNVH DDRDRPS QVWDSSRDQAVIof SEQ ID NO: 19 (SEQ ID NO: 10) (SEQ ID NO: 11) (SEQ ID NO: 12)

Sequences RSV F protein full length sequence subgroup A (SEQ ID NO: 13)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAVKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSNRSV F protein B1 full length sequence (SEQ ID NO: 15)MELLIHRLSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYFSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAANNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEINREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITTIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSKSEQ ID NO: 14 (fibritin) GYIPEAPRDGQAYVRKDGEWVLLSTFL RSV F protein CL57-v224 full length sequence (SEQ ID NO: 20)MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIE  LSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANNRARRELPRFMNYTLNNTKNNNVTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNVGKSTTNIMITTIIIVIIVILLLLIAVGLFLYCKARSTPVTLSKDQLSGINNIAFSNRSV F, N67I, S215P, D486N, and 357K and 371Y (SEQ ID NO: 21)MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKEIKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAEKCKVQSNRVFCDTMYSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSNEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL CR9501 heavy chain (SEQ ID NO: 16):QVQLVQSGPGLVKPSQTLALTCNVSGASINSDNYYWTWIRQRPGGGLEWIGHISYTGNTYYTPSLKSRLSMSLETSQSQFSLRLTSVTAADSAVYFCAACGAYVLISNCGWFDSWGQGTQVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCCR9501 light chain (SEQ ID NO: 17):EIVMTQSPSSLSASIGDRVTITCQASQDISTYLNWYQQKPGQAPRLLIYGASNLETGVPSRFTGSGYGTDFSVTISSLQPEDIATYYCQQYQYLPYTFAPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECCR9502 heavy chain (SEQ ID NO: 18):EVQLLQSGAELKKPGASVKISCKTSGFTFSGHTIAWVRQAPGQGLEWMGWVSTNNG NTEYAQKIQGRVTMTMDTSTSTVYMELRSLTSDDTAVYFCAREWLVMGGFAFDHWGQGTLLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCCR9502 light chain (SEQ ID NO: 19):QSVLTQASSVSVAPGQTARITCGANNIGSQNVHWYQQKPGQAPVLVVYDDRDRPSGIPDRFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSRDQAVIFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTIAPTECS

The invention claimed is:
 1. A recombinant pre-fusion respiratorysyncytial virus (RSV) Fusion (F) protein comprising a mutation of theamino acid residue at position 215, a mutation of the amino acid residueat position 357, and a mutation of the amino acid residue at position371, wherein the amino acid positions correspond to the numbering of theamino acid sequence of SEQ ID NO:
 13. 2. The pre-fusion RSV F proteinaccording to claim 1, comprising a mutation of the amino acid residueSer at position 215 to Pro (S215P), a mutation of the amino acid residueThr at position 357 to Lys (T357K) and a mutation of the amino acidresidue Asn at position 371 to Tyr (N371Y).
 3. The pre-fusion RSV Fprotein according to claim 1, further comprising at least one mutationselected from the group consisting of: a) a mutation of the amino acidresidue at position 67; and b) a mutation of the amino acid residue atposition
 486. 4. The pre-fusion RSV F protein according to claim 3,wherein the at least one further mutation is selected from the groupconsisting of: c) a mutation of the amino acid residue Asn/Thr atposition 67 to Ile; and d) a mutation of the amino acid residue Asp atposition 486 to Asn.
 5. The pre-fusion RSV F protein according to claim1, wherein the protein comprises at least one epitope that is specificto the pre-fusion conformation F protein, wherein the at least oneepitope is recognized by a pre-fusion specific monoclonal antibody,comprising a heavy chain CDR1 region of SEQ ID NO: 1, a heavy chain CDR2region of SEQ ID NO: 2, a heavy chain CDR3 region of SEQ ID NO: 3 and alight chain CDR1 region of SEQ ID NO: 4, a light chain CDR2 region ofSEQ ID NO: 5, and a light chain CDR3 region of SEQ ID NO: 6 and/or apre-fusion specific monoclonal antibody, comprising a heavy chain CDR1region of SEQ ID NO: 7, a heavy chain CDR2 region of SEQ ID NO: 8, aheavy chain CDR3 region of SEQ ID NO: 9 and a light chain CDR1 region ofSEQ ID NO: 10, a light chain CDR2 region of SEQ ID NO: 67, and a lightchain CDR3 region of SEQ ID NO:
 11. 6. The pre-fusion RSV F proteinaccording to claim 1, wherein the protein is trimeric.
 7. The pre-fusionRSV F protein according to claim 1, comprising a truncated F1 domain anda heterologous trimerization domain linked to the truncated F1 domain.8. The pre-fusion RSV F protein according to claim 7, wherein theheterologous trimerization domain comprises the amino acid sequenceGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 14).
 9. The pre-fusion RSV Fprotein according to claim 7, wherein the trimerization domain is linkedto amino acid residue 513 of the RSV F protein.
 10. The pre-fusion RSV Fprotein according to claim 1, wherein the RSV F protein comprises theamino acid sequence of SEQ ID NO:
 21. 11. A nucleic acid moleculeencoding the pre-fusion RSV F protein according to claim
 1. 12. Thenucleic acid molecule according to claim 11, wherein the nucleic acidmolecule has been codon-optimized for expression in mammalian cells. 13.A vector comprising the nucleic acid molecule according to claim
 11. 14.A composition comprising a pre-fusion RSV F protein according toclaim
 1. 15. A method of inducing an immune response against RSV Fprotein in a subject in need thereof, the method comprisingadministering to the subject the pre-fusion RSV F protein according toclaim
 1. 16. A vaccine comprising the pre-fusion RSV F protein accordingto claim
 1. 17. A method of prophylaxis and/or treatment of RSVinfection in a subject in need thereof, the method comprisingadministering to the subject the pre-fusion RSV F protein according toclaim
 1. 18. A host cell comprising the nucleic acid molecule accordingto claim
 11. 19. A method of preparing the pre-fusion RSV F proteinaccording to claim 1, comprising growing a host cell comprising anucleic acid molecule encoding the pre-fusion RSV F protein underconditions for expression of the pre-fusion RSV F protein.
 20. A nucleicacid molecule encoding the pre-fusion RSV F protein according to claim10.
 21. A composition comprising a pre-fusion RSV F protein according toclaim
 10. 22. A composition comprising the nucleic acid moleculeaccording to claim
 11. 23. A composition comprising the nucleic acidmolecule according to claim
 20. 24. A method of inducing an immuneresponse against RSV F protein in a subject in need thereof, the methodcomprising administering to the subject the pre-fusion RSV F proteinaccording to claim
 10. 25. A method of inducing an immune responseagainst RSV F protein in a subject in need thereof, the methodcomprising administering to the subject the nucleic acid moleculeaccording to claim
 11. 26. A method of inducing an immune responseagainst RSV F protein in a subject in need thereof, the methodcomprising administering to the subject the nucleic acid moleculeaccording to claim
 20. 27. A vaccine comprising the pre-fusion RSV Fprotein according to claim
 10. 28. A vaccine comprising the nucleic acidmolecule according to claim
 11. 29. A vaccine comprising the nucleicacid molecule according to claim
 20. 30. A method of prophylaxis and/ortreatment of RSV infection in a subject in need thereof, the methodcomprising administering to the subject the pre-fusion RSV F proteinaccording to claim
 10. 31. A method of prophylaxis and/or treatment ofRSV infection in a subject in need thereof, the method comprisingadministering to the subject the nucleic acid molecule according toclaim
 11. 32. A method of prophylaxis and/or treatment of RSV infectionin a subject in need thereof, the method comprising administering to thesubject the nucleic acid molecule according to claim 20.